Acinetobacter baumannii multivalent subunit recombinant vaccine as well as preparation method and application thereof

Abstract

The invention discloses an acinetobacter baumannii multivalent subunit recombinant vaccine as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The recombinant vaccine comprises eight antigen proteins including BamA, Bap, Ata, NucAb, OmpA, BauA, PKF and Hcp, the purity of each antigen protein is greater than 90%, and the antigen proteins are mixed with an aluminum hydroxide gel adjuvant (the aluminum content is 1.3-1.5 mg / mL) according to the volume ratio of 1: 1. The preparation method comprises the following steps: cloning and constructing an antigen gene expression vector, inducing soluble expression, carrying out affinity / ion exchange chromatography purification, and standing and adsorbing an antigen-adjuvant compound. According to the invention, by integrating eight antigen proteins, namely BamA, Bap, Ata, NucAb, OmpA, BauA, PKF and Hcp, multiple pathogenic pathways of the acinetobacter baumannii are covered. Compared with the defect that an existing single antigen vaccine is prone to failure due to strain variation, the multivalent antigen can induce an organism to generate broad-spectrum antibody response through the synergistic effect, and the high genetic heterogeneity of the acinetobacter baumannii is effectively dealt with.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a multivalent subunit recombinant vaccine of Acinetobacter baumannii, its preparation method, and its application. Background Technology

[0002] Acinetobacter baumannii, a Gram-negative drug-resistant bacterium, has become the leading threat to nosocomial infections worldwide due to its extreme environmental tolerance and multidrug resistance mechanisms (especially MDRAB strains with carbapenem resistance rates >50%). The mortality rate of ventilator-associated pneumonia, neonatal sepsis, and central nervous system infections caused by it is as high as 40-60%, thus it has been listed as a "critical priority pathogen" by the World Health Organization (WHO). Currently, traditional antibiotic treatment faces a dual challenge: on the one hand, the sensitivity of drug-resistant bacteria to antibiotics continues to decline, leading to treatment failure; on the other hand, the development cycle of novel antimicrobial drugs is long and costly, making it difficult to meet clinical needs. Against this backdrop, vaccine development has three urgent strategic implications: directly reducing antibiotic use by preventing infection and cutting off the evolutionary selection pressure of drug resistance; establishing a pre-emptive protective barrier for immunocompromised populations (such as premature infants and burn patients) to avoid the risk of treatment non-response; and utilizing herd immunity to block biofilm-mediated nosocomial transmission chains, curbing the spread of drug-resistant bacteria at the source.

[0003] In existing technologies, single-antigen vaccines are prone to failure due to bacterial strain mutations, while multivalent subunit recombinant vaccines can achieve broad-spectrum and highly effective immune protection by integrating antigens covering different pathogenic pathways of bacteria. This invention targets the high genetic heterogeneity and multiple pathogenic mechanisms of Acinetobacter baumannii (including adhesion and colonization, immune escape, biofilm formation, nutrient depletion, and virulence secretion), and designs a multivalent vaccine containing multiple conserved antigens, aiming to overcome existing drug resistance barriers. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multivalent subunit recombinant vaccine for Acinetobacter baumannii, which induces a highly efficient humoral immune response in the body by combining multiple conserved antigens, thereby achieving effective prevention and control of Acinetobacter baumannii infection.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A multivalent subunit recombinant vaccine for Acinetobacter baumannii includes eight antigen proteins: BamA, Bap, Ata, NucAb, OmpA, BauA, PKF, and Hcp, with each antigen protein having a purity >90%.

[0007] Furthermore, the amino acid sequence of the BamA antigen protein is shown in SEQ ID NO:1;

[0008] The amino acid sequence of the Bap antigen protein is shown in SEQ ID NO:2;

[0009] The amino acid sequence of the Ata antigen protein is shown in SEQ ID NO:3;

[0010] The amino acid sequence of the NucAb antigen protein is shown in SEQ ID NO:4;

[0011] The amino acid sequence of the OmpA antigen protein is shown in SEQ ID NO:5;

[0012] The amino acid sequence of the BauA antigen protein is shown in SEQ ID NO:6;

[0013] The amino acid sequence of the PKF antigen protein is shown in SEQ ID NO:7;

[0014] The amino acid sequence of the Hcp antigen protein is shown in SEQ ID NO:8.

[0015] Furthermore, it also includes aluminum hydroxide gel adjuvant and PBS buffer. Eight antigen proteins, namely BamA, Bap, Ata, NucAb, OmpA, BauA, PKF and Hcp, are mixed in equal mass and dissolved in PBS buffer to prepare an antigen solution. The aluminum hydroxide gel adjuvant is mixed with the antigen solution at a volume ratio of 1:1, and the final concentration of total antigen after mixing is 0.32 mg / mL.

[0016] Furthermore, the aluminum content in the aluminum hydroxide gel adjuvant is 1.3-1.5 mg / mL.

[0017] The present invention also provides a method for preparing the above-mentioned Acinetobacter baumannii multivalent subunit recombinant vaccine, comprising the following steps:

[0018] S1, gene expression vectors for the antigen proteins BamA, Bap, Ata, NucAb, OmpA, BauA, PKF and Hcp were cloned and constructed, and transformed into Escherichia coli expression strains.

[0019] S2 induces the expression of soluble antigen protein, and the expression effect is verified by SDS-PAGE electrophoresis;

[0020] S3, the target antigen protein is purified by affinity chromatography or ion exchange chromatography, and the antigen protein with a purity >90% is obtained after dialysis and desalting.

[0021] S4 involves mixing eight antigen proteins, mixing them with adjuvants in a specific ratio, and allowing them to stand and adsorb, forming an antigen-adjuvant complex, which is the Acinetobacter baumannii multivalent subunit recombinant vaccine.

[0022] Furthermore, the expression vector mentioned in step S1 is one of pET28a(+), pGEX-6p-1, and pET32a(+), and the Escherichia coli expression strain is E. coli Rosetta(DE3) or E. coli BL21(DE3).

[0023] Furthermore, the conditions for inducing expression in step S2 are: final IPTG concentration of 0.2-0.6 mM, induction temperature of 16℃, and culture time of 12-16 h.

[0024] Furthermore, the affinity chromatography in step S3 uses a Ni-NTA column or a GST column, and the ion exchange chromatography uses a Capto... TM Q or Capto TM S ImpAct ion column.

[0025] Furthermore, in step S4, the adsorption time is 30 minutes and the temperature is room temperature.

[0026] Furthermore, the present invention also provides the use of the above-mentioned Acinetobacter baumannii multivalent subunit recombinant vaccine in the preparation of a drug for preventing Acinetobacter baumannii infection.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This invention integrates eight antigen proteins—BamA, Bap, Ata, NucAb, OmpA, BauA, PKF, and Hcp—to achieve full-cycle interception from adhesion and colonization to immune evasion and virulence output. BamA (>97% conservation ensures broad-spectrum coverage), Bap / Ata (disrupts biofilm resistance barriers), OmpA / PKF (dual-pathway blocking of immune escape: OmpA inhibits host recognition escape, and PKF neutralizes complement resistance), NucAb (high-safety adhesion blocking), BauA (iron deprivation strategy), and Hcp (targeting the virulence secretion system). Compared to the shortcomings of existing single-antigen vaccines, which are prone to failure due to strain mutations, multivalent antigens can induce a broad-spectrum antibody response through synergistic effects, effectively addressing the high genetic heterogeneity of Acinetobacter baumannii. Experimental data show that combined immunization with the eight antigens can achieve a 100% protection rate, far superior to single-antigen groups and quadruple-antigen groups.

[0029] (2) In this invention, aluminum hydroxide gel adjuvant and antigen solution are mixed at a 1:1 volume ratio (aluminum content 1.3-1.5 mg / mL), and a stable antigen-adjuvant complex is formed through an adsorption process that allows the mixture to stand at room temperature for 30 minutes. This ratio can effectively enhance antigen presentation efficiency and stimulate a strong humoral immune response. In the experiment, the antibody titer of immunized mice reached 1:51200, which can ensure the immunogenicity of the vaccine.

[0030] (3) In actual use, the total antigen concentration in the antigen-adjuvant complex of the recombinant antigen provided by the present invention is 0.32 mg / mL. The total antigen concentration is low, which can ensure the immune effect while avoiding immune tolerance or toxic reactions caused by excessive antigen. It is suitable for safe vaccination of immune vulnerable populations (such as premature infants and burn patients).

[0031] (4) In the process of preparing recombinant vaccines, this invention uses expression vectors such as pET28a(+) and pGEX-6p-1 in combination with Rosetta(DE3) and BL21(DE3) Escherichia coli strains to achieve efficient expression of soluble antigen proteins. High-purity antigen proteins can then be obtained through simple separation and purification steps. The processes for preparing both the antigen proteins and the recombinant vaccine (antigen-adjuvant complex) are relatively simple and suitable for large-scale production.

[0032] (5) The recombinant vaccine provided by this invention can be used to prepare drugs for preventing Acinetobacter baumannii infection, providing a novel preventive measure for nosocomial infections (such as ventilator-associated pneumonia and neonatal sepsis) caused by multidrug-resistant Acinetobacter baumannii (MDRAB). By reducing antibiotic use through active immunization, interrupting the evolutionary selection pressure of drug resistance, and simultaneously utilizing herd immunity to block the biofilm-mediated transmission chain, it has significant strategic importance for global public health security. Attached Figure Description

[0033] Figure 1 Figure showing the purification results of BamA antigen protein:

[0034] Figure 2 The image shows the purification results of the Bap antigen protein.

[0035] Figure 3 The image shows the purification results of the Ata antigen protein;

[0036] Figure 4 The image shows the purification results of the NucAb antigen protein.

[0037] Figure 5 The image shows the purification results of the OmpA antigen protein.

[0038] Figure 6 The image shows the purification results of the BauA antigen protein.

[0039] Figure 7 The image shows the purification results of the PKF antigen protein.

[0040] Figure 8 The image shows the purification results of the Hcp antigen protein;

[0041] Figure 9 This is a graph showing the intensity of antibody responses in immunized mice to recombinant antigens (eight antigens).

[0042] Figure 10 Survival rate curves for different bacterial concentrations when constructing a sepsis model;

[0043] Figure 11 The efficacy of immunoprotection was evaluated for single antigen groups, quadruple antigen groups, and eight-antigen combination groups. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0045] The experimental strains, plasmids, experimental mice, various reagents and instruments used in this embodiment were all purchased from the market. Among them, pET28a(+) plasmid, pET30a(+) plasmid, pgex-6p-1 plasmid, pgex-6p-2 plasmid, and E. coli TOP10 were purchased from Wuhan Jinkairui Biotechnology Co., Ltd., E. coli Rosetta (DE3) and E. coli BL21 (DE3) were purchased from Beijing TransGen Biotechnology Co., Ltd., Acinetobacter baumannii AB5075 was from the Western Theater Command General Hospital, and BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The sources of the main reagents are shown in Table 1.

[0046] Table 1. Sources of main reagents

[0047] Types of reagents source Kanamycin Shanghai McLean Biochemical Technology Co., Ltd. Chloramphenicol Shanghai McLean Biochemical Technology Co., Ltd. Ampicillin Shanghai McLean Biochemical Technology Co., Ltd. IPTG (Isopropyl-β-D-thiogalactoside) biosharp 12.5% ​​PAGE Gel Rapid Preparation Kit Shanghai Yamei Biomedical Technology Co., Ltd. <![CDATA[Capto TM Q ion column]]> Cytiva <![CDATA[Capto TM S ImpAct ion column]]> Cytiva HisSep Ni-NTA Agaroses Resin Yisheng Biotechnology (Shanghai) Co., Ltd. GST-tag Purification Resin Shanghai Beyotime Biotechnology Co., Ltd. Goat anti-mouse IgG Abcam PBS phosphate buffer Beijing Solarbio Technology Co., Ltd. Protein molecular weight standards Wuhan Saiweier Biotechnology Co., Ltd. BamHⅠ restriction endonuclease Takara XhoⅠ restriction endonuclease Takara

[0048] Example 1

[0049] This embodiment provides a multivalent subunit recombinant vaccine against Acinetobacter baumannii, which includes an adjuvant and eight antigen proteins: BamA, Bap, Ata, NucAb, OmpA, BauA, PKF, and Hcp. The method for preparing these eight antigen proteins is as follows:

[0050] I. Preparation of BamA antigen protein

[0051] 1. Cloning and construction of the BamA gene

[0052] (1) By comparing sequences with NCBI, it was found that the amino acid sequence of BamA was highly conserved among different strains of Acinetobacter baumannii. Based on the amino acid sequence of BamA, the gene fragment of BamA was obtained. The amino acid sequence of BamA is shown in SEQ ID NO:1.

[0053] (2) The target gene was synthesized in its entirety and ligated into the expression vector pET28a(+) (with a His tag) via BamHI and XhoI restriction sites. The plasmid sequencing results were identical to the target gene sequence, and analysis confirmed the absence of amino acid mutations. The recombinant plasmid pET28a(+)-BamA was extracted from the recombinant pET28a(+) / BamA / E. coli TOP10 strain.

[0054] (3) The recombinant plasmid was transformed into the expression strain E. coli Rosetta(DE3) to obtain recombinant Escherichia coli pET28a(+) / BamA / E. coli Rosetta(DE3) that can express BamA.

[0055] 2. Verify the soluble expression of recombinant BamA protein.

[0056] (1) Activation of bacteria: Take the candidate antigen protein expression bacteria pET28a(+) / BamA / E.coliRosetta(DE3) stored at -80℃ and activate the bacteria by inoculation at a volume ratio of 1:1000, that is, add 10μL of glycerol bacterial solution to 10mL of LB medium containing Kanamycin and Chl chloramphenicol resistance, and incubate overnight at 37℃ and 220rpm for 16h.

[0057] (2) Secondary activation induction: The bacteria were inoculated at a 1:100 volume ratio for secondary activation. Specifically, 200 μL of overnight bacterial culture was added to 20 mL of LB medium containing Kana and Chl resistance, and incubated at 37°C and 220 rpm for 5-6 hours. Secondary activation was continued until the OD value reached... 600 When the concentration is 0.6, add IPTG to a final concentration of 0.6 mM, and incubate overnight on a shaker at 16°C and 150 rpm. (Preparation of IPTG solution: Dissolve 2.38 g of IPTG in 10 mL of Grade I water, and filter through a 0.22 μm sterile filter after complete dissolution).

[0058] (3) Take out the bacterial culture after induction and centrifuge at 4℃ and 6000rpm for 15min. Discard the supernatant, add 1-2mL of equilibration solution to resuspend and mix well, sonicate for 15min to break the bacteria, and collect the lysed solution. Centrifuge at 4℃ and 12000rpm for 15min to separate the supernatant and precipitate.

[0059] (4) Processing the supernatant and precipitate: Take 20 μL of supernatant and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min; add 30 μL of equilibration buffer to the precipitate to resuspend the bacterial cells, take 20 μL of the resuspended bacterial solution and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min.

[0060] (5) Pour 12.5% ​​separating gel into the gel casting plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 minutes. Pour off the top layer of distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 minutes. Take 10 μL of the processed supernatant and precipitate respectively for SDS-PAGE electrophoresis. Electrophore at 80V for 30 minutes, then adjust to 180V and electrophore for 1-2 hours. After electrophoresis, remove the gel, place it in staining solution and shake to stain, then place it in water and shake to destain. Observe the results under the imaging system. BamA is a soluble protein.

[0061] 3. Acquisition, isolation, and purification of BamA antigen protein

[0062] (1) Scale-up culture to obtain bacterial culture: Take 20 μL of pET28a(+) / BamA / E.coli Rosetta(DE3) bacterial culture stored at 4℃ and add it to 20 mL of LB medium containing Kana and Chl resistance. Incubate overnight at 37℃ and 200 rpm for the first activation. Take 20 mL of the first-activated bacterial culture and add it to 2000 mL of LB medium containing Kana and Chl resistance for the second activation. Incubate at 37℃ for 5-6 h. Add 0.6 mM IPTG and incubate overnight at 16℃ and 150 rpm for induction. Collect the bacterial cells by centrifugation at 4℃ and 6000 rpm for 15 min. After resuspending the bacterial cells in 50 mL of equilibration buffer PBS, perform high-pressure lysis of the bacterial culture (pressure 700-800 bar).

[0063] (2) Protein binding to packing material: After lysis, the solution was centrifuged at 12000 rpm for 15 min at 4℃. The supernatant was collected and purified using a Ni column. The Ni column was a Ni-NTA affinity chromatography column, and the packing material was HisSep Ni-NTA Agarose Resin. The packing material volume was 100 mL per 100 g of lysed bacterial wet weight. Impurities were washed with 5 column volumes of elution buffer containing 20 mM imidazole and 5 column volumes of elution buffer containing 50 mM imidazole, and samples were taken. BamA protein was eluted with 5 column volumes of elution buffer containing 100 mM imidazole, 200 mM imidazole, and 300 mM imidazole, respectively, and samples were taken for SDS-PAGE electrophoresis. Finally, the packing material was washed with elution buffer containing 500 mM imidazole, rinsed with pure water, and stored in 20% ethanol. Each gradient sampling label was used for electrophoretic detection (results are shown in the figure). Figure 1 As shown in the figure, the final result was a BamA protein with a molecular weight of 92 kDa and high purity.

[0064] Figure 1 The lanes in the middle are:

[0065] Lane M. Protein molecular weight standard; Lane 1. Complete lysate; Lane 2. Lysate precipitate; Lane 3. Lysate supernatant; Lane 4. Breakthrough buffer; Lane 5. Elution buffer 1 containing 20 mM imidazole; Lane 6. Elution buffer 2 containing 20 mM imidazole; Lane 7. Elution buffer 1 containing 50 mM imidazole; Lane 8. Elution buffer 2 containing 50 mM imidazole; Lane 9. Elution buffer 1 containing 100 mM imidazole; Lane 10. Elution buffer 2 containing 100 mM imidazole; Lane 11. Elution buffer 1 containing 200 mM imidazole; Lane 12. Elution buffer 2 containing 200 mM imidazole; Lane 13. Elution buffer 1 containing 300 mM imidazole; Lane 14. Elution buffer 2 containing 300 mM imidazole.

[0066] To prepare the elution buffer containing 20 mM imidazole: Take 48 mL of equilibration PBS and 2 mL of PBS elution buffer containing 500 mM imidazole; elution buffer 1 containing 20 mM imidazole is the eluent collected after eluting 2.5 column volumes, and elution buffer 2 containing 20 mM imidazole is the eluent collected after eluting again with the 20 mM imidazole-containing elution buffer. To prepare the elution buffer containing 50 mM imidazole: Take 45 mL of equilibration PBS and 5 mL of PBS elution buffer containing 500 mM imidazole; elution buffer 1 containing 50 mM imidazole is the eluent collected after eluting 2.5 column volumes, and elution buffer 2 containing 50 mM imidazole is the eluent collected after eluting again with the 20 mM imidazole-containing elution buffer. And so on.

[0067] II. Preparation of Bap antigen protein

[0068] The method for preparing Bap antigen protein is the same as that for preparing BamA antigen protein, with the only difference being the separation and purification steps, as follows:

[0069] The amino acid sequence of Bap is shown in SEQ ID NO:2. Based on the amino acid sequence, the Bap gene fragment was obtained and recombinant *E. coli* pET28a(+) / Bap / E. coli Rosetta(DE3) was first cloned and constructed. After verifying the solubility of the target antigen protein, the culture was amplified to obtain bacterial suspension. The bacterial suspension was autoclaved and centrifuged at 4°C and 12,000 rpm for 15 min. The supernatant was collected and purified using a Ni column. Impurities were washed with 5 column volumes of elution buffer containing 20 mM imidazole and 5 column volumes of elution buffer containing 50 mM imidazole. Samples were then collected. Subsequently, Bap protein was eluted sequentially with 5 column volumes of elution buffer containing 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole. Samples were then subjected to SDS-PAGE electrophoresis. Collect the eluent containing 200 mM imidazole and dialyze overnight at 4°C (dialysis buffer: 50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5). The eluent also contains 50 mM Hepes, 100 mM KCl, 10% glycerol, and pH 7.5.

[0070] Using Capto TM S ImpAct ion exchange column purification, collecting the flow-through liquid and labeling it as the permeate. Elution conditions: washing with a solution of 50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5; gradient elution with a solution of 50 mM Hepes, 200-500 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5. Preferably, the KCl concentrations for gradient elution are 200 mM, 300 mM, 400 mM, and 500 mM. Approximately two column volumes are collected for each gradient (the exact number of column volumes collected depends on the UV absorption peak of the chromatography instrument; if two column volumes of eluent are collected, the first column volume is eluent 1, and the second column volume is eluent 2). Samples from each gradient are labeled and analyzed by electrophoresis (results are shown in the figure). Figure 2 As shown in the figure, the final Bap protein with a molecular weight of 46 kDa and high purity (purity > 95%) was obtained.

[0071] Figure 2 The lanes in the middle are:

[0072] Lane M. Protein molecular weight standard; Lane 1. Before loading; Lane 2. Breakthrough buffer; Lane 3. Elution buffer 1 containing 200 mM KCl; Lane 4. Elution buffer 2 containing 200 mM KCl; Lane 5. Elution buffer 1 containing 300 mM KCl; Lane 6. Elution buffer 2 containing 300 mM KCl; Lane 7. Elution buffer containing 400 mM KCl; Lane 8. Elution buffer containing 500 mM KCl.

[0073] III. Preparation of Ata antigen protein

[0074] 1. Cloning and construction of the Ata gene

[0075] The same cloning method as the BamA gene was used, but the expression vector selected in this step was pgex-6p-1. The amino acid sequence of Ata is shown in SEQ ID NO:3. Finally, recombinant Escherichia coli pgex-6p-1 / Ata / E. coli Rosetta(DE3) that can express Ata was obtained.

[0076] 2. Verify the soluble expression of Ata protein

[0077] (1) Activation of bacteria: Take the candidate antigen protein expression bacteria pgex-6p-1 / Ata / E.coli Rosetta (DE3) stored at -80℃ and activate them by inoculation at a ratio of 1:100, that is, add 100μL of glycerol bacterial solution to 10mL of LB medium containing resistance to Amp (ampicillin) and Chl (chloramphenicol) and incubate at 37℃ and 250rpm for 4h.

[0078] (2) Secondary activation induction: Inoculate the bacteria at a 1:50 ratio for secondary activation. Take 200 μL of overnight bacterial culture and add it to 10 mL of LB medium containing Amp and Chl resistance. Incubate at 37℃ and 220 rpm for 5-6 h. Activate to OD. 600 When the concentration is 0.6, add IPTG to a final concentration of 0.2 mM, and incubate overnight on a shaker at 16°C and 150 rpm. (Preparation of IPTG solution: Dissolve 2.38 g of IPTG in 10 mL of Grade I water, and filter through a 0.22 μm sterile filter after complete dissolution).

[0079] (3) Take out the bacterial culture after induction and centrifuge at 4℃ and 6000rpm for 15min. Discard the supernatant, add 1-2mL of equilibration solution to resuspend and mix well, sonicate for 15min to break the bacteria, and collect the lysed solution. Centrifuge at 4℃ and 12000rpm for 15min to separate the supernatant and precipitate.

[0080] (4) Processing the supernatant and precipitate: Take 20 μL of supernatant and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min; add 30 μL of equilibration buffer to the precipitate to resuspend the bacterial cells, take 20 μL of the resuspended bacterial solution and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min.

[0081] (5) Pour 12.5% ​​separating gel into the gel casting plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 minutes. Pour off the top layer of distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 minutes. Take 10 μL of the processed supernatant and precipitate respectively for SDS-PAGE electrophoresis. Electrophore at 80V for 30 minutes, then adjust to 180V and electrophore for 1-2 hours. After electrophoresis, remove the gel, place it in staining solution and shake to stain, then place it in water and shake to destain. Observe the results under the imaging system. Ata is a soluble protein.

[0082] 3. Preparation of Ata antigens

[0083] (1) Scale-up culture to obtain bacterial culture: Take 200 μL of pgex-6p-1 / Ata / E.coli Rosetta (DE3) bacterial culture stored in a 4℃ refrigerator and add it to 20 mL of LB medium containing Amp and Chl resistance. Incubate at 37℃ and 250 rpm for 4 h for the first activation. Then take 40 mL of the first activated bacterial culture and add it to 2000 mL of LB medium containing Amp and Chl resistance for the second activation. Incubate at 37℃ and 180 rpm for 5-6 h until the OD value is about 0.6. Add 0.2 mM IPTG and incubate overnight at 16℃ and 150 rpm in a shaker. Collect the bacterial cells by centrifugation at 4℃ and 6000 rpm for 15 min. Resuspend each 2.3g of bacterial cells in 60mL of equilibration buffer (50mM Tris-HCl, 200mM NaCl, 2mM TCEP, 0.01% Triton X-100, pH 8.0), then autoclave the bacterial solution (700-800 bar). After autoclaving, centrifuge at 4℃ and 12000rpm for 30min and collect the supernatant.

[0084] (2) Protein binding to the packing material: The supernatant was purified using a GST column. Preferably, the packing material for the GST column was GST-tag Purification Resin. 10 mL of GST packing material was placed in the chromatography column, and the packing material was washed three times with 25 mL of pure water. Then, the column volume was equilibrated with equilibration buffer. The lysed supernatant was added to the chromatography column and mixed with the GST packing material. The mixture was then placed on a rotary shaker at 4 °C for 2 h to bind the protein.

[0085] Wash the impurities three times with equilibration buffer, 25 mL each time, and take samples from each wash. Reequilibrate three times with enzyme digestion buffer (50 mM HEPES, 200 mM NaCl, 20% glycerol, 2 mM TCEP, 1 mM EDTA, 0.05% Tween-20, pH 7.0), 25 mL each time. After the liquid has drained, add 10 mL of enzyme digestion buffer to the column, mix well, and take samples from each column. Then add 1.4 mL of 0.9 mg / mL PP enzyme, mix well, and incubate overnight at 4°C for enzyme digestion. After overnight digestion, collect the permeate; filter the permeate and take samples from each column. Elute twice with elution buffer (50 mM HEPES, 100 mM NaCl, 20% glycerol, 2 mM TCEP, 0.01% Tween-20, pH 6.5), 25 mL each time, and take samples from each eluent. The protein isolation and purification results in this step are as follows: Figure 3 As shown in the figure (lanes are: lane M, protein molecular weight standard; lane 1, lysed whole solution; lane 2, lysed supernatant; lane 3, lysed precipitate; lane 4, permeate; lanes 5-7, elution buffer for other proteins; lane 8, before enzyme digestion; lane 9, after enzyme digestion; lane 10, filtration after enzyme digestion; lane 11, elution buffer 1; lane 12, elution buffer 2), the target protein with a molecular weight of 27 kDa was finally obtained.

[0086] IV. Preparation of NucAb antigen protein

[0087] The method for preparing NucAb antigen protein is the same as that for preparing BamA antigen protein; the difference lies in the separation and purification steps, as detailed below:

[0088] The amino acid sequence of NucAb is shown in SEQ ID NO:4. Based on the amino acid sequence, the NucAb gene fragment was obtained and first cloned to construct recombinant *E. coli* pET28a(+) / NucAb / E. coli Rosetta(DE3). After verifying the solubility of the target antigen protein, the culture was scaled up to obtain bacterial suspension. The bacterial suspension was autoclaved and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected and purified using a Ni column. The Ni column was a Ni-NTA affinity chromatography column, and the packing material was HisSep Ni-NTA garose Resin. The packing material volume was 100 mL per 100 g of lysed bacterial cells (wet weight). Five column volumes of elution buffer containing 20 mM imidazole and five column volumes of elution buffer containing 50 mM imidazole were used to wash away contaminating proteins before sampling. The collected samples were sequentially eluted with a gradient of 5 column volumes of elution buffer containing 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole to remove NucAb protein. SDS-PAGE electrophoresis was then performed on the eluent. The eluent containing 100 mM imidazole was collected and dialyzed overnight at 4°C (dialysis buffer: 50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5). In addition to different concentrations of imidazole, the eluent also contained 50 mM Hepes, 100 mM KCl, 10% glycerol, and had a pH of 7.5.

[0089] Using Capto TM Q-ion column purification; Capto was rinsed with pure water and equilibration buffer. TM The Q ion column was run three times, and protein solution that had been dialyzed overnight was added. Linear elution was performed using solution A (50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5) and solution B (50 mM Hepes, 500 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5). Approximately two column volumes were collected for each gradient (the exact amount depends on the UV absorption peak of the chromatography instrument). Each gradient sample was labeled and analyzed by electrophoresis. The results are shown below. Figure 4 As shown in the figure (lanes are: lane M. protein molecular weight standard; lane 1. before loading; lane 2. breakthrough buffer; lane 3. 0-20% B elution buffer 1; lane 4. 0-20% B elution buffer 2; lane 5. 20-40% B elution buffer 1; lane 6. 20-40% B elution buffer 2; lane 7. 20-40% B elution buffer 3; lane 8. 500mM KCl elution buffer), the final result was a NucAb protein with a molecular weight of 44kDa and high purity.

[0090] V. Preparation of OmpA antigen protein

[0091] 1. Cloning and construction of the OmpA gene

[0092] The same cloning method as the BamA gene was used, but the expression vector selected in this step was pET32a(+). The amino acid sequence of OmpA is shown in SEQ ID NO:5. Finally, recombinant Escherichia coli pET32a(+) / OmpA / E.coli Rosetta(DE3) that can express OmpA was obtained.

[0093] 2. Verify the soluble expression of OmpA protein.

[0094] (1) Activation of bacteria: Take the candidate antigen protein expression bacteria pET32a(+) / OmpA / E.coli Rosetta(DE3) stored at -80℃ and activate them by inoculation at a ratio of 1:1000, that is, add 10 μL of glycerol bacterial solution to 10 mL of LB medium containing resistance to Amp (ampicillin) and Chl (chloramphenicol), and incubate overnight at 37℃ and 220 rpm for 16 h.

[0095] (2) Secondary activation induction: A 1:100 inoculation ratio was used for secondary activation. 200 μL of overnight bacterial culture was added to 20 mL of LB medium containing Amp and Chl resistance, and incubated at 37°C and 220 rpm for 5-6 hours. Secondary activation was continued until the OD value reached... 600 When the concentration is 0.6, add IPTG to a final concentration of 0.5 mM, and incubate overnight at 16°C and 150 rpm on a shaker. (Preparation of IPTG solution: Dissolve 2.38 g of IPTG in 10 mL of Grade I water, and filter through a 0.22 μm sterile filter after complete dissolution).

[0096] (3) Take out the bacterial culture after induction and centrifuge at 4℃ and 6000rpm for 15min. Discard the supernatant, add 1-2mL of equilibration solution to resuspend and mix well, sonicate for 15min to break the bacteria, and collect the lysed solution. Centrifuge at 4℃ and 12000rpm for 15min to separate the supernatant and precipitate.

[0097] (4) Processing the supernatant and precipitate: Take 20 μL of supernatant and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min; add 30 μL of equilibration buffer to the precipitate to resuspend the bacterial cells, take 20 μL of the resuspended bacterial solution and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min.

[0098] (5) Pour 12.5% ​​separating gel into the gel casting plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 minutes. Pour off the top layer of distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 minutes. Take 10 μL of the processed supernatant and precipitate respectively for SDS-PAGE electrophoresis. Electrophore at 80V for 30 minutes, then adjust to 180V and electrophore for 1-2 hours. After electrophoresis, remove the gel, place it in staining solution and shake to stain, then place it in water and shake to destain. Observe the results under the imaging system. OmpA is a soluble protein.

[0099] 3. Preparation of OmpA antigen

[0100] (1) Scale-up culture to obtain bacterial culture: Take 20 μL of pET32a(+) / OmpA / E. coli Rosetta(DE3) bacterial culture stored at 4℃ and add it to 20 mL of LB medium containing Amp and Chl resistance. Incubate overnight at 37℃ and 200 rpm for the first activation. Take 20 mL of the first activated bacterial culture and add it to 2000 mL of LB medium containing Amp and Chl resistance for the second activation. Incubate at 37℃ for 5-6 h. Add 0.5 mM IPTG and incubate overnight at 16℃ and 150 rpm for induction. Centrifuge at 4℃ and 6000 rpm for 15 min to collect the bacterial cells. Add 50 mL of equilibration buffer (50 mM Hepes, 100 mM KCl, 10% glycerol, pH 7.5) to resuspend the bacterial cells. Perform high-pressure lysis of the bacterial culture (pressure 700-800 bar) and collect the supernatant.

[0101] (2) Protein binding to packing material: After lysis, the solution was centrifuged at 12000 rpm for 15 min at 4℃. The supernatant was collected and purified using a Ni column. Preferably, a Ni-NTA affinity chromatography column was selected, with HisSep Ni-NTA Agarose Resin as the packing material. The packing material volume was 100 mL per 100 g of lysed bacterial wet weight. Impurities were washed with 5 column volumes of elution buffer containing 20 mM imidazole and 5 column volumes of elution buffer containing 50 mM imidazole, and then a sample was taken. OmpA protein was eluted using a gradient of 5 column volumes of elution buffer containing 100 mM, 200 mM, 300 mM, and 500 mM imidazole. Finally, the target protein eluted with 500 mM imidazole was collected, diluted 1:2 with dialysis buffer (50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5), and dialyzed overnight at 4°C.

[0102] (3) Rinse Capto with pure water and balancing solution. TMOn a S ImpAct ion exchange column, protein solution that has been dialyzed overnight is added. The solution is washed with solution A (50 mM Hepes, 150 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5). Then, a gradient elution is performed with solution B (50 mM Hepes, 200-500 mM KCl, 1 mM DTT, 10% glycerol, pH 7.5). The purified protein is collected, approximately two column volumes per gradient (the exact volume depends on the UV absorption peak of the chromatography instrument). Samples from each gradient are labeled and analyzed by electrophoresis. Results are shown below. Figure 5 As shown in the figure (lanes are: lane M. protein molecular weight standard; lane 1. lysis supernatant; lane 2. permeate; lane 3. elution buffer containing 20 mM imidazole; lane 4. elution buffer 1 containing 50 mM imidazole; lane 5. elution buffer 2 containing 50 mM imidazole; lane 6. elution buffer containing 100 mM imidazole; lane 7. elution buffer containing 200 mM imidazole; lane 8. elution buffer containing 300 mM imidazole; lane 9. elution buffer 1 containing 500 mM imidazole; lane 10. elution buffer 2 containing 500 mM imidazole), because the vector plasmid pET32a(+) itself carries the TrxA tag, the final obtained OmpA protein has a molecular weight of 58 kDa and high purity.

[0103] VI. Preparation of BauA antigen protein

[0104] 1. Cloning and construction of the BauA gene

[0105] The same cloning method as the BamA gene was used, but the expression vector selected in this step was pGEX-6P-2, the expression strain was E. coli BL21, and the amino acid sequence of BauA is shown in SEQ ID NO:6. Finally, recombinant E. coli pGEX-6P-2 / BauA / E. coli BL21 that can express BauA was obtained.

[0106] 2. Verify the soluble expression of BauA protein.

[0107] (1) Activation of bacteria: Take the candidate antigen protein expression bacteria pGEX-6P-2 / BauA / E.coliBL21 stored at -80℃ and activate it by inoculation at a ratio of 1:1000, that is, add 10μL of glycerol bacterial solution to 10mL of LB medium containing Amp resistance and incubate overnight at 37℃ and 200rpm for 16h.

[0108] (2) Secondary activation induction: Inoculate the bacteria at a 1:100 ratio for secondary activation. Take 200 μL of overnight bacterial culture and add it to 20 mL of LB medium containing Amp resistance. Incubate at 37℃ and 220 rpm for 5-6 h. Activate to OD... 600When the concentration is 0.6, add IPTG to a final concentration of 0.2 mM, and incubate overnight on a shaker at 16°C and 150 rpm. (Preparation of IPTG solution: Dissolve 2.38 g of IPTG in 10 mL of Grade I water until completely dissolved, and filter through a 0.22 μm sterile filter.)

[0109] (3) Take out the bacterial culture after induction and centrifuge at 4℃ and 6000rpm for 15min. Discard the supernatant, add 1-2mL PBS to resuspend and mix well, sonicate for 15min, and collect the lysed culture. Centrifuge at 4℃ and 12000rpm for 15min to separate the supernatant and precipitate.

[0110] (4) Processing the supernatant and precipitate: Take 20 μL of supernatant and add 5 μL of 5× protein loading buffer, boil for 5 min, and then centrifuge at 14000 rpm for 3 min; add 30 μL of PBS to the precipitate to resuspend the bacterial cells, take 20 μL of the resuspended bacterial solution and add 5 μL of 5× protein loading buffer, boil for 5 min, and then centrifuge at 14000 rpm for 3 min.

[0111] (5) Pour 10% separating gel into the gel casting plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 minutes. Pour off the top layer of distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 minutes. Take 10 μL of the processed supernatant and precipitate respectively for SDS-PAGE electrophoresis. Electrophore at 80V for 30 minutes, then adjust to 180V and electrophore for 1-2 hours. After electrophoresis, remove the gel, place it in Coomassie Brilliant Blue staining solution and shake to stain, then place it in destaining solution and shake to destain. Observe the results under the imaging system. BauA is a soluble protein.

[0112] 3. Preparation of BauA antigen

[0113] (1) Scale-up culture to obtain bacterial culture: 200 μL of pGEX-6P-2 / BauA / E.coli BL21 bacterial culture stored at 4℃ was added to 20 mL of LB medium containing Amp resistance for primary activation. After incubation at 37℃ and 200 rpm for 5-6 h, 20 mL of the primary activated bacterial culture was added to 2000 mL of LB medium containing Amp resistance for secondary activation. After incubation at 37℃ for 5-6 h, IPTG was added to a final concentration of 0.2 mM and incubated overnight at 16℃ and 150 rpm. The bacterial cells were collected by centrifugation at 4℃ and 12000 rpm for 15 min. The bacterial cells were resuspended in 50 mL of PBS and subjected to high-pressure disruption (pressure 700-800 bar). After centrifugation at 4℃ and 12000 rpm for 30 min, the supernatant was collected and 5 mL of GST packing was added. The mixture was incubated on a side-shaking shaker at 4℃ for 5-6 h. 40 μL of the flow-through was retained for SDS-PAGE gel electrophoresis. Wash three times with 50 mL PBST-NaCl, and retain 40 μL of the washing buffer for SDS-PAGE gel electrophoresis. Equilibrate three times with 50 mL PBS, and resuspend in 50 mL PBS.

[0114] (2) Using an enzyme digestion method, the target protein and GST tag were separated to obtain the BauA target protein. 2 mL of PP enzyme was added to the remaining GST packing material containing the bound protein, and the mixture was digested overnight at 4°C. After elution with 50 mL of PBS, the eluent was retained for SDS-PAGE gel electrophoresis. The digestion and eluent were ultrafiltered using 30 kDa ultrafiltration tubes and labeled as Concentration 1 and Concentration 2, respectively. The results were observed using an imaging system. Figure 6 As shown, the molecular weight of the enzyme-digested BauA protein is 78 kDa, which is consistent with the expected protein molecular weight.

[0115] VII. Preparation of PKF antigen protein

[0116] 1. Cloning and construction of the PKF gene

[0117] The same cloning method as the BamA gene was used, but the expression vector selected in this step was pgex-6p-2. The amino acid sequence of PKF is shown in SEQ ID NO:7. Finally, recombinant Escherichia coli pgex-6p-2 / PKF / E.coli Rosetta(DE3) that can express PKF was obtained.

[0118] 2. Verify the soluble expression of PKF protein.

[0119] (1) Activation of bacteria: Take the candidate antigen protein expression bacteria pgex-6p-2 / PKF / E.coli Rosetta (DE3) stored at -80℃ and activate them by inoculation at a ratio of 1:1000, that is, add 10 μL of glycerol bacterial solution to 10 mL of LB medium containing resistance to Amp (ampicillin) and Chl (chloramphenicol) and incubate overnight at 37℃ and 220 rpm.

[0120] (2) Secondary activation induction: A 1:100 inoculation ratio was used for secondary activation. 100 μL of overnight bacterial culture was added to 10 mL of LB medium containing Amp and Chl resistance, and incubated at 37℃ and 220 rpm for 5-6 h. Secondary activation was continued until OD... 600 When the concentration is 0.6, add IPTG to a final concentration of 0.2 mM, and incubate overnight on a shaker at 16°C and 120 rpm. (Preparation of IPTG solution: Dissolve 2.38 g of IPTG in 10 mL of Grade I water, and filter through a 0.22 μm sterile filter after complete dissolution).

[0121] (3) Take out the bacterial culture after induction and centrifuge at 4℃ and 6000rpm for 15min. Discard the supernatant, add 1-2mL of equilibration solution to resuspend and mix well, sonicate for 15min to break the bacteria, and collect the lysed solution. Centrifuge at 4℃ and 12000rpm for 15min to separate the supernatant and precipitate.

[0122] (4) Processing the supernatant and precipitate: Take 20 μL of supernatant and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min; add 30 μL of equilibration buffer to the precipitate to resuspend the bacterial cells, take 20 μL of the resuspended bacterial solution and add 5 μL of 5× protein loading buffer, boil for 10 min, and then centrifuge at 14000 rpm for 3 min.

[0123] (5) Pour 12.5% ​​separating gel into the gel casting plate, add distilled water to flatten the gel, and let it solidify at room temperature for 30 minutes. Pour off the top layer of distilled water, then pour in the stacking gel, immediately insert the comb, and let it solidify at room temperature for 30 minutes. Take 10 μL of the processed supernatant and precipitate respectively for SDS-PAGE electrophoresis. Electrophore at 80V for 30 minutes, then adjust to 180V and electrophore for 1-2 hours. After electrophoresis, remove the gel, place it in staining solution and shake to stain, then place it in water and shake to destain. Observe the results under the imaging system. PKF is a soluble protein.

[0124] 3. Preparation of PKF antigen

[0125] (1) Scale-up culture to obtain protein: Take 20 μL of pgex-6p-2 / PKF / E.coli Rosetta (DE3) bacterial culture stored in a 4℃ refrigerator and add it to 20 mL of LB medium containing Amp and Chl resistance. Incubate overnight at 37℃ and 220 rpm for the first activation. Take 20 mL of the first activated bacterial culture and add it to 2000 mL of LB medium containing Amp and Chl resistance for the second activation. Incubate at 37℃ and 180 rpm for 5-6 h until the OD value is about 0.6. Add 0.2 mM IPTG and incubate overnight at 16℃ and 120 rpm in a shaker. Collect the bacterial cells by centrifugation at 4℃ and 6000 rpm for 15 min. After resuspending the bacterial cells in 50 mL of equilibration buffer (50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 2 mM TCEP, 0.01% Triton X-100, pH 8.0), the bacterial solution was autoclaved (700-800 bar). After autoclaving, the solution was centrifuged at 4 °C and 12,000 rpm for 30 min, and the supernatant was collected.

[0126] (2) Protein binding to the packing material: The supernatant was collected and purified using a GST column. Preferably, the packing material for the GST column was GST-tag Purification Resin. 10 mL of the packing material was placed in the chromatography column and washed three times with 25 mL of pure water. The column volume was then equilibrated with equilibration buffer to a final volume of 3. The lysed supernatant was added to the chromatography column and mixed with the GST beads. The column was then placed in a rotating shaker at 4°C for 2 hours. The equilibration buffer was used to wash away any remaining protein three times, 25 mL each time. The enzyme digestion buffer (50 mM Tris-HCl, 500 mM NaCl, 15% glycerol, 5 mM TCEP, 1 mM EDTA, 0.02% Triton X-100, pH 8.0) was used to reequilibrate twice, 25 mL each time. After the liquid had drained, 20 mL of the enzyme digestion buffer was added to the column, mixed, and the sample was collected. Then, 2 mL of 2.0 mg / mL PP enzyme was added, mixed, and the column was incubated at 4°C overnight for enzyme digestion. After overnight enzyme digestion, the permeate was collected; the permeate was filtered and sampled again; elution was performed four times with 10 mL of elution buffer (50 mM Tris-HCl, 150 mM NaCl, 5% glycerol, 2 mM TCEP, pH 8.0), and samples were collected from each elution. The separation and purification results are as follows: Figure 7 As shown in the figure (lanes are: Lane M. Protein molecular weight standard; Lane 1. Complete lysis solution; Lane 2. Cycle precipitate; Lane 3. Cycle supernatant; Lane 4. Breakthrough solution; Lane 5. Rebalancing 1; Lane 6. Rebalancing 2; Lane 7. Before enzyme digestion; Lane 8. Breakthrough solution after enzyme digestion; Lane 9. Filtration of breakthrough solution after enzyme digestion; Lane 10. Wash 1; Lane 11. Wash 2; Lane 12. Wash 3; Lane 13. Wash 4), the target protein with a molecular weight of 47 kDa was finally obtained.

[0127] 8. Preparation of Hcp antigen protein

[0128] 1. Cloning and construction of the Hcp gene

[0129] The same cloning method as the BamA gene was used, but the expression vector selected in this step was pGEX-6P-1, the expression strain was E. coli BL21, and the amino acid sequence of Hcp is shown in SEQ ID NO:8. Finally, recombinant E. coli pGEX-6P-1 / Hcp / E. coli BL21 that can express Hcp was obtained.

[0130] 2. Verify the soluble expression of Hcp protein

[0131] The soluble expression of Hcp protein was verified using a method for verifying the soluble expression of BauA protein, with the only difference being that the concentration of the separating gel was 12.5%. The results confirmed that Hcp is a soluble protein.

[0132] 3. Preparation of Hcp antigen

[0133] (1) Protein Acquisition by Scale-up Culture: 200 μL of pGEX-6P-1 / Hcp / E.coli BL21 bacterial culture stored at 4℃ was added to 20 mL of LB medium containing Amp resistance for primary activation. After incubation at 37℃ for 5-6 h at 200 rpm, 20 mL of the primary activated bacterial culture was added to 2000 mL of LB medium containing Amp resistance for secondary activation. After incubation at 37℃ for 5-6 h, 0.2 mM IPTG was added and the culture was incubated overnight at 150 rpm in a shaker at 16℃. The bacterial cells were collected by centrifugation at 12000 rpm for 15 min at 4℃. The bacterial cells were resuspended in 50 mL of PBS, and the bacterial culture was subjected to high-pressure lysis (700-800 bar). After centrifugation at 12000 rpm for 30 min at 4℃, the supernatant was collected and 5 mL of LST packing material was added. The culture was incubated on a side-shaking shaker at room temperature for 2 h. 40 μL of the flow-through was retained for SDS-PAGE gel electrophoresis. Wash twice with 25 mL PBST, then equilibrate twice with 50 mL PBS. Resuspend the beads in 10 mL PBS in the chromatography column and perform SDS-PAGE gel electrophoresis.

[0134] (2) Using an enzyme digestion method, the target protein and GST tag were separated to obtain the Hcp target protein. 1 mL of PP enzyme was added to the remaining GST packing material containing the bound protein, and the mixture was digested overnight at 4°C. The permeate was collected, filtered through a 0.22 μM sterile filter, and samples were taken for SDS-PAGE gel electrophoresis. 25 mL of PBS was added for elution twice, and the eluent was retained. Samples were then taken for SDS-PAGE gel electrophoresis. The results were observed under an imaging system. The results are as follows: Figure 8As shown in the figure (lanes are: lane 1, lysis supernatant; lane 2, lysis precipitate; lane 3, PBST wash 1; lane 4, PBST wash 2; lane 5, PBS equilibration 1; lane 6, PBS equilibration 2; lane 7, permeate 1; lane 8, permeate 2; lane 9, permeate after enzyme digestion; lane 10, filtration of permeate after enzyme digestion; lane 11, PBS elution 1; lane 12, PBS elution 2), the molecular weight of the Hcp protein after enzyme digestion is 19 kDa, which is consistent with the expected protein molecular weight.

[0135] Example 2

[0136] The antibody titers after combined immunization with the eight antigen proteins prepared in Example 1 were evaluated as follows:

[0137] 1. Laboratory animals

[0138] Preparation: Select 6-8 week old SPF grade BALB / c female mice (weighing 18-22g), with 5 mice in each of the experimental and control groups. They were kept in an SPF environment for 7 days for acclimatization, with free access to food and water, and their health status was observed daily.

[0139] Antigen and adjuvant preparation: The experimental group antigen contained eight antigen proteins (purity >90%): BamA, Bap, Ata, NucAb, OmpA, BauA, PKF, and Hcp. Each antigen was administered at a dose of 20 μg. The total antigen was dissolved in sterile PBS to prepare a 250 μL solution. The adjuvant was aluminum hydroxide gel (aluminum content 1.3-1.5 mg / mL), mixed at a volume ratio of antigen solution to adjuvant of 1:1 (i.e., 250 μL antigen solution + 250 μL aluminum hydroxide). After vortexing and allowing to stand at room temperature for 30 min, an antigen-adjuvant complex was formed (final total antigen concentration 0.32 mg / mL). The control group used an equal volume of PBS instead of the antigen solution, and the aluminum hydroxide adjuvant was mixed using the same method.

[0140] Immunization schedule: Three intramuscular injections were administered on day 0 (primary immunization), day 14, and day 21 (booster immunization). Each mouse was injected with 100 μL of antigen-adjuvant complex (containing 50 μL of antigen / PBS solution and 50 μL of aluminum hydroxide).

[0141] Serum collection and processing: Blood was collected via tail vein on day 28 post-immunization, with ≤100μL of whole blood collected each time. After the blood was allowed to stand at room temperature for 30 minutes, it was centrifuged at 3000rpm for 10 minutes at 4℃ to separate the serum. The serum was then aliquoted and stored at -80℃ protected from light to avoid repeated freeze-thaw cycles.

[0142] Antibody titers were evaluated using the ELISA method.

[0143] Coating antigen: Eight antigen proteins were mixed (2 μg / mL, pH 9.6 carbonate buffer, 4°C overnight).

[0144] Sealing: After washing the plate, seal with 3% BSA, 200 μL / well, at 37°C for 1 hour.

[0145] Serum dilution gradient: Serum from immunized mice was serially diluted starting at 1:100. Simultaneously, serum from control mice was used as a negative control, and PBST was used as a blank control. (Add 100 μL of PBST to all wells of the ELISA plate except for rows 1 and 12. Add 200 μL of diluted immunized mouse serum to the wells in row 1, then transfer 100 μL from row 1 to row 2 and mix 10 times using a multi-channel pipette. Repeat this process, performing 2-fold serial dilutions until row 11. Finally, discard the 100 μL solution in row 11.) Incubate at 37°C for 1 h.

[0146] Secondary antibody addition: After washing the plate, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody and incubate at a dilution ratio of 1:10000 for 45 min at 37°C.

[0147] Color development and termination: Wash the plate again, add 100 μL of TMB substrate, and incubate at 37°C in the dark for 10 min. After the color development is complete, terminate the reaction with 50 μL of stop solution.

[0148] Titer determination: After the reaction was terminated, the OD value of each well was measured using a microplate reader within 15 minutes, and the optical density at 450 nm was recorded. The antibody titer was determined by the highest dilution with an OD value ≥ 2.1 times that of the negative control. This process allows for a systematic assessment of the antibody response strength of immunized mice to the antigen protein (results are shown in the figure). Figure 9 (As shown in the figure). It can be seen that the mice immunized with the combined eight antigens showed a good level of antibody humoral response, the antigen immunization effect was in line with the experimental expectations, and the antibody titer reached 1:51200.

[0149] 2. Construction of a sepsis model

[0150] (1) Take Acinetobacter baumannii AB5075 from the -80℃ freezer and thaw it on ice. Dip the inoculation loop into the bacterial solution and streak it in three zones on the MH plate. Place it in a 37℃ incubator and incubate it upside down overnight.

[0151] (2) The next day, pick a single colony and inoculate it into 5 mL of MH culture medium and incubate overnight in a shaker at 37°C and 220 rpm.

[0152] (3) Collect the bacterial culture, centrifuge at 12000 rpm for 1 min, and resuspend in 2 mL of sterile PBS.

[0153] (4) Detection of OD 600 Adjust the absorbance to 1, take 10 μL of bacterial culture and add it to 90 μL of MH medium for serial dilution, and then serially dilute to 10 μL. -9 The diluted solution was spread onto MH plates and incubated upside down at 37°C overnight. The next day, the number of colonies on the plates was counted and the OD was calculated. 600 =1 CFU / mL. Calculation formula: CFU / mL = Colony count × 1 / (Dilution factor × 0.1).

[0154] (5) Adjust the bacterial concentration to 1×10 9 cfu / mL, 7.5×10 8 cfu / mL, 5×10 8 cfu / mL, 2.5×10 8 cfu / mL. Twenty female BALB / c mice were randomly divided into four groups and challenged with the bacterial solution via intraperitoneal injection, with each mouse receiving 100 μL of the solution. Survival rates were recorded for 7 consecutive days, and the survival curves are shown below. Figure 10 As shown in the figure, the highest concentration is 1×10⁻⁶. 9 The bacterial solution at CFU / mL caused the death of all mice; the two intermediate concentrations had a lethality of 80%; and the lowest concentration of 2.5 × 10⁻⁶ CFU / mL resulted in death. 8 Mice with cfu / mL achieved a 100% survival rate. In this initial pharmacodynamic experiment, excessive challenge could lead to rapid onset of disease, high mortality, and difficulty in observing drug efficacy. Therefore, a challenge dose with a mortality rate of 60%-90% was most suitable. For survival rate evaluation, a dose of 5 × 10⁻⁶ was selected. 8 cfu / mL is used as the lethal challenge dose.

[0155] 3. Evaluation of the protective efficacy of combined immunization with eight antigens

[0156] Seventy SPF-grade BALB / c female mice (6-8 weeks old) were randomly divided into 7 groups (n=10 per group). The specific grouping and treatment are as follows:

[0157] Blank control group: PBS and aluminum hydroxide adjuvant mixed at a 1:1 ratio;

[0158] Single antigen group (3 groups): 20 μg of single antigen (BamA / OmpA / BauA) was dissolved in PBS to prepare an antigen solution, and then mixed with adjuvant at a ratio of 1:1;

[0159] The quadruple antigen group (2 groups): Group A (BamA, Bap, Ata, NucAb) and Group B (OmpA, BauA, PKF, Hcp). Each group of antigens (20 μg each) was mixed and dissolved in PBS to prepare an antigen solution, which was then mixed with adjuvant at a 1:1 ratio.

[0160] Eight-antigen combination group: Eight antigens (20 μg each) were mixed and dissolved in PBS to prepare an antigen solution, which was then mixed with adjuvant at a 1:1 ratio. The final antigen concentration after mixing in each group was 0.32 mg / mL.

[0161] After vortexing and mixing the above groups, the mixture was allowed to stand at room temperature for 30 minutes to adsorb and form an antigen-adjuvant complex. Each mouse was injected intramuscularly with 100 μL of the antigen-adjuvant complex. Mice in each group received booster immunizations on days 14 and 21 after the initial immunization, for a total of three immunizations. On day 35 post-immunization, AB5075 (5 × 10⁻⁶) was injected via the tail vein. 8 The virus challenge experiment was conducted using CFU / ml. Survival rates were recorded daily for 7 days after challenge, and the survival rate curves for each group are shown below. Figure 11 As shown in the figure, the combined immunization with eight antigens achieves a protective efficiency of 100%.

[0162] The amino acid sequences of the eight antigen proteins in this invention are shown below:

[0163] SEQ ID NO:1 (Amino acid sequence of BamA)

[0164] ADDFVVRDIRVNGLVRLTPANVYTMLPINSGDRVNEPMIAEAIRTLYATGLFDDIKASKENDTLVFNVIERPIISKLEFKGNKLIPKEALEQGLKKMGIAEGEVFKKSALQTIETELEQQYTQQGRYDADVTVDTVARPNNRVELKINFNEGTPAKVFDINVIGNTVFKDSEIKQAFAVKESGWASVVTRNDRYAREKMAASLEALRAMYLNKGYINFNINNSQLNISEDKKHIFIEVAVDEGSQFKFGQTKFLGDALYKPEELQALKIYKDGDTYSQEKVNAVKQLLLRKYGNAGYYFADVNIVPQINNETGVVDLNYYVNPGQQVTVRRINFTGNSKTSDEVLRREMRQMEGALASNEKIDLSKVRLERTGFFKTVDIKPARIPNSPDQVDLNVNVEEQHSGTTTLAVGYSQSGGITFQAGLSQTNFMGTGNRVAIDLSRSETQDYYNLSVTDPYFTIDGVSRGYNVYYRKTKLNDDYNVNNYVTDSFGGSLSFGYPIDENQSLSASVGVDNTKVTTGPYVSTYVRDYLLANGGKATSKGTYCPTDANGNSQYDTEKGECKVPEETYDNAFEGEFFTYNLNLGWSYNTLNRPIFPTSGMSHRVGLEIGLPGSDVDYQKVTYDTQAFFPIGSTGFVLRGYGKLGYGNDLPFYKNFYAGGYGSVRGYDNSTLGPKYPSVNLQETKQNDSSPEEVGGNALVQFGTELVLPMPFKGDWTRQVRPVLFAEGGQVFDTKCNIDNSVYGNKGMKINGQTITDVRKYCEDNYGFDLGNLRYSVGVGVTWITMIGPLSLSYAFPLNDKPGDETKEIQFEIGRTF

[0165] SEQ ID NO:2 (Amino acid sequence of Bap)

[0166] GSMKNFLTKKMYATRALFFAVGVMALNVSSIVISTTHAATEQKMDNLSTKLSLIFADKPIEASLFSPQFLEQVPITQIQKIVDDLKVSLGALKNINVSNGSGTIDFEKGELPVSISLNEQGQISTLWFSAPHFKTISLDEMVKGLHENAIGKTSLLVIVDNKPVVVENDKTPMAVGSTFKLLVLKAYEDAIKKGELKRETIVSLKEKNRSLPTGVLQNLPANTPVNLELLAQLMIQISDNTATDSLIEILKKPRIEALSPRNSPLLTTRELFQLIDPSNEKLRNKFKKGTKSARLEALAELDKLPLPSVSSIGKLATWQDAEWYMSANEICPLLESVQDAPALNSSLNPLFKNLNWQKIGFKGGSEYGVINFSVIGKTQKGHKVCAVFTANGNEPQPESKLAILFTGILQAVDSMNHLE

[0167] SEQ ID NO:3 (Amino acid sequence of Ata)

[0168] KGTNLKNVADGKVAEGSKDAVNGGQLWNVQNQVDKNSNDIKNIQNNIDNISNGKAGLVQQQKPNGEITVGKDTGGTSINMAGKEGDRVVQGVKDGEIKAGSNQAVNGGQIHKISESIKNSIGGNTTIDPKDGSITTNNIGGTGKNNINDAIGTLNQSNQELGNKITNLGDQLQQVFYDTNKRIDDVEKKANAGIAAAMALENAPFVAGKYTYAVGAAYHGGENAVGVTLRKTSDNGRWSITGGVAAASQGEPSVRVGISGVIN

[0169] SEQ ID NO:4 (Amino acid sequence of NucAb)

[0170] GSMQLSIFEHYKQLLKNAKKYKIPPREKTFFDTAIRNHYENPTTELLEFFLNPTESHDLGDLFWKGFCDVLQQEASLSKLDLGNIVKLEREYATHQGNRIDLWIETDTCFILLEAKIYHHQNNPFQDYIQFAQSKNQSKNKQIVGVILSIAGKSEKKGWLGLSYQQIVNSIRPYLAEQMLANPMNKWNLFAREFLLHLDSYYRIKNLDMNRVQFILDHYKEIEELQRLRTSTISEVVDSLSQQLNEMIDGYESENKYESWGGIRFYNKAWGNKSNTTLLIKQEDGQTVIKVITYILNLSLELEEEAFSILGTQTDSRYLDQKVENYRRGTERWLCIYWRSPENNLTAITDLLFEKVKLLDTIERTLKLE

[0171] SEQ ID NO:5 (Amino acid sequence of OmpA)

[0172] GSMKLSRIALATMLVAAPLAAANAGVTVTPLLLGYTFQDSQHNNGGKDGNLTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGASAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYDFDGVNRGTRGTSEEGTLGNAGVGAFWRLNDALSLRTEARATYNADEEFWNYTALAGLNVVLGGHLKPAAPVVEVAPVEPTPVAPQPQELTEDLNMELRVFFDTNKSNIKDQYKPEIAKVAEKLSEYPNATARIEGHTDNTGPRKLNERLSLARANSVKSALVNEYNVDASRLSTQGFAWDQPIADNKTKEGRAMNRRVFATITGSRTVVVQPGQEAAAPAAAQLE

[0173] SEQ ID NO:6 (Amino acid sequence of BauA)

[0174] MALRLGYALGTVFVLCASNTYAAVIDNSTKTLEQQTAQTNVAALPAITVKAE

[0175] QDDTYAGGQVATSSNVGFLGSKKFLDTPFNTISYTDKYIEDKQAKDITEVIAA

[0176] TDPSIYTNGASGGWSENYYIRGYASSTNDMSMNGLFGITPFYRTSPEMFGRVE

[0177] VLKGPSALLNGMPPAGSVGGTVNLVTKYAADEPFARLTTTYMSDAQFGGHV

[0178] DVGRRFGENKEFGVRINGMYRDGDAAVNDQSKESRLFSLGLDWQGENARVF

[0179] VDAYDALDHVDGVTRGVNVSTAVGIPKPPKADTLLSPDWGSVETKDKGAMI

[0180] RGEYDFSDQLMAYAAYGQSTTEYKYNGASAGTITSSTGTLSSTLGQLAFDVD

[0181] KKSADAGFKGKFETGSVKHQWVANATYYNHTQDDYGYRIIPGFSDPVITNIY

[0182] DPNPNWGPKPEFTPPFLFHSTLSTSSFGLADTLSFAQDKVQLTLGLRHQTVKA

[0183] TSSVNTLPENAKSATTPGVALLIKATDKISVYANYIEGLTKGDQAPATASNPGEI

[0184] FPPQKTKQQELGLKVDLGTFAHTLSAFEITKPSSYLDPSKLVNNLPTFVSDGE

[0185] QRNRGIEWSFFGSPIEHVRLMGGFTYLDPELTKTKSGGNDGHTAVAVPKNQA

[0186] KLGAEWDTQVAQGTLTLSGNINAVSKQYINAENTLSVPGRTLLDVGARYSTK

[0187] VEDHPVTFRANIYNLTNKAYWAQPQLTNLALGAPRTYMLSVSYDF

[0188] SEQ ID NO:7 (Amino acid sequence of PKF)

[0189] AVDFSNLVEQVSPAVVSVNVVKKMTQDELLQQQVPEILKRFFGNQVIIPQQQGPQEKTAYGSAFFISKDGYLLTNHHVIENASRISITLNDRREIDATVVGSDERTDV ALLKVNGTNYPALRVGNVDRLRVGEPVLAIGSPFGFDYSASAGIVSAKSRNMSGETSVPFIQTDVALNPGNSGGPLFNQNGEVVGVNSRIFSGTGGYMGLSFSIPIDVA MDVAEQLKTKGKVTRSYLGVMMQDIDRNLADAYKLPKPEGALITQVSPNSPAQKAGLRAGDVILKLNGASVLRTSDLLYALNKVQPNQTVQFEVLRDDKTRNISATLA TAPDETPATGNQASASKGPVLGMSIRDLAVPEKNALGIKGGIYVQDVRRGGLASLSNIIPGDVIIQVNNTQILNSQDFAKVVSNLPKNTVARVGIIRQGQRAMLGLRIQ

[0190] SEQ ID NO:8 (Amino acid sequence of Hcp)

[0191] GSMKDIYVEFRGKYKVDGESRDSEHKGWLEVNSWSHNIRQPKSATSSSVGGHTAERVEHSDMVFVKDLDATSPKLWEACSAGYTFDEVQIDFYRANGDKRIKYLQIKLKHVLVSSVTPTVNEEGVPTEAFGLKYAAVEWTYNQQDINGTAKGAVTKKWSLSNNTASYAA

[0192] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A multivalent subunit recombinant vaccine against Acinetobacter baumannii, characterized in that, It includes eight antigen proteins: BamA, Bap, Ata, NucAb, OmpA, BauA, PKF, and Hcp, and the purity of each antigen protein is >90%.

2. The Acinetobacter baumannii multivalent subunit recombinant vaccine according to claim 1, characterized in that, The amino acid sequence of the BamA antigen protein is shown in SEQ ID NO:1; The amino acid sequence of the Bap antigen protein is shown in SEQ ID NO:2; The amino acid sequence of the Ata antigen protein is shown in SEQ ID NO:3; The amino acid sequence of the NucAb antigen protein is shown in SEQ ID NO:4; The amino acid sequence of the OmpA antigen protein is shown in SEQ ID NO:5; The amino acid sequence of the BauA antigen protein is shown in SEQ ID NO:6; The amino acid sequence of the PKF antigen protein is shown in SEQ ID NO:7; The amino acid sequence of the Hcp antigen protein is shown in SEQ ID NO:

8.

3. The Acinetobacter baumannii multivalent subunit recombinant vaccine according to claim 2, characterized in that, It also includes aluminum hydroxide gel adjuvant and PBS buffer. Eight antigen proteins, namely BamA, Bap, Ata, NucAb, OmpA, BauA, PKF and Hcp, are mixed in equal mass and dissolved in PBS buffer to prepare an antigen solution. The aluminum hydroxide gel adjuvant and the antigen solution are mixed at a volume ratio of 1:

1. The final concentration of total antigen after mixing is 0.32 mg / mL.

4. The Acinetobacter baumannii multivalent subunit recombinant vaccine according to claim 3, characterized in that, The aluminum content in the aluminum hydroxide gel adjuvant is 1.3-1.5 mg / mL.

5. A method for preparing the Acinetobacter baumannii multivalent subunit recombinant vaccine as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1, gene expression vectors for the antigen proteins BamA, Bap, Ata, NucAb, OmpA, BauA, PKF and Hcp were cloned and constructed, and transformed into Escherichia coli expression strains. S2 induces the expression of soluble antigen protein, and the expression effect is verified by SDS-PAGE electrophoresis; S3, the target antigen protein is purified by affinity chromatography or ion exchange chromatography, and the antigen protein with a purity >90% is obtained after dialysis and desalting. S4 involves mixing eight antigen proteins, mixing them with adjuvants in a specific ratio, and allowing them to stand and adsorb, forming an antigen-adjuvant complex, which is the Acinetobacter baumannii multivalent subunit recombinant vaccine.

6. The method for preparing a multivalent subunit recombinant vaccine of Acinetobacter baumannii according to claim 5, characterized in that, The expression vector mentioned in step S1 is one of pET28a(+), pGEX-6p-1, and pET32a(+), and the Escherichia coli expression strain is E. coli Rosetta(DE3) or E. coli BL21(DE3).

7. The method for preparing a multivalent subunit recombinant vaccine of Acinetobacter baumannii according to claim 6, characterized in that, The conditions for induction of expression in step S2 are: final IPTG concentration of 0.2-0.6 mM, induction temperature of 16℃, and culture time of 12-16 h.

8. The method for preparing a multivalent subunit recombinant vaccine of Acinetobacter baumannii according to claim 7, characterized in that, The affinity chromatography in step S3 uses a Ni-NTA column or a GST column, and the ion exchange chromatography uses a Capto... TM Q or Capto TM SImpAct ion column.

9. The method for preparing a multivalent subunit recombinant vaccine of Acinetobacter baumannii according to claim 8, characterized in that, In step S4, the adsorption time is 30 minutes and the temperature is room temperature.

10. The use of the Acinetobacter baumannii multivalent subunit recombinant vaccine according to any one of claims 1 to 4 in the preparation of a medicament for the prevention of Acinetobacter baumannii infection.

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